Enhancement of a biotechnological platform for the purification and delivery of a human papillomavirus supercoiled plasmid DNA vaccine.
N Biotechnol
; 59: 1-9, 2020 Nov 25.
Article
en En
| MEDLINE
| ID: mdl-32622863
New biotechnological strategies are being explored, aimed at rapid and economic manufacture of large quantities of DNA vaccines with the required purity for therapeutic applications, as well as their correct delivery as biopharmaceuticals to target cells. This report describes the purification of supercoiled (sc) HPV-16 E6/E7 plasmid DNA (pDNA) vaccine from a bacterial lysate, using an arginine-based monolith, presenting a spacer arm in its configuration. To enhance the performance of the purification process, monolith modification with the spacer arm can improve accessibility of the arginine ligand. By using a low NaCl concentration at pH 7.0, a condition to eliminate the RNA impurity directly in the flow through was established. The pH increase to 7.5 allowed the elimination of non-functional pDNA isoforms, the sc pDNA being recovered by increasing the ionic strength. As well as a binding capacity of 2.53 mg/mL obtained with a pre-purified sc pDNA sample, the column also purified sc pDNA from high lysate loading, with capacities above 1 mg/mL. Due to the sample displacement phenomena, non-functional pDNA isoforms were eliminated throughout column loading, favoring the degree of purity of final sc pDNA of 93.3%-98.5%. Thereafter, purified sc pDNA was successfully encapsulated into CaCO3-gelatin nano-complexes. Delivery of the pDNA-carriers to THP-1 cells was assessed through pDNA cellular uptake evaluation and correct E6 expression was verified by mRNA and protein detection. A biotechnological platform was established for sc pDNA purification and delivery to dendritic cells, stimulating further in vivo studies.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Represoras
/
Biotecnología
/
ADN Superhelicoidal
/
Proteínas Oncogénicas Virales
/
Vacunas de ADN
/
Proteínas E7 de Papillomavirus
/
Alphapapillomavirus
Límite:
Humans
Idioma:
En
Revista:
N Biotechnol
Asunto de la revista:
BIOLOGIA MOLECULAR
/
ENGENHARIA BIOMEDICA
Año:
2020
Tipo del documento:
Article
País de afiliación:
Portugal
Pais de publicación:
Países Bajos